Impact of inoculum density of Fusarium oxysporum f. sp. zingiberi on symptomatic appearances and yield of ginger (Zingiber officinale Roscoe)

Ginger (Zingiber officinale Roscoe) is an important horticultural crop valued for its medicinal and culinary properties. Fusarium yellows, caused by the ascomycete fungus Fusarium oxysporum f. sp. zingiberi (Foz), is a devastating soil-borne disease of ginger. It has curtailed ginger production in Australia and around the world, leading to significant economic losses. An integrated approach is required to manage soil-borne diseases such as those caused by Foz. However, little is known about the influence of Foz inoculum on disease severity. This study aimed to establish a minimum threshold level of spores per gram of soil required for plant infection and to develop and evaluate a pot inoculation method for screening large numbers of plants in a controlled environment. To achieve this, the dominant Australian ginger cultivar Canton was inoculated with 101, 103, 105, 106 and 107 microconidia g−1 soil. The inoculum density was positively associated with leaf and stem yellows, and rhizome discolouration, and negatively associated with root length and rhizome weight. The lowest threshold required for plant infection was 101 microconidia g−1 soil, which may provide an important basis for outbreaks of Foz in the field. This finding adds significantly to our knowledge of the impact of soil health on ginger production, thereby contributing to the integrated management of Foz. When used at a high dose, this method can facilitate reliable and accurate screening of Foz-susceptible ginger genotypes in a controlled environment.


INTRODUCTION
Ginger (Zingiber officinale Roscoe) is an important horticultural crop valued for its medicinal and culinary properties.It has traditionally been used to reduce nausea, provide remedies for influenza and the common cold, and aid digestion [1].In food preparations, ginger has been widely used as a cooking spice, and it is also consumed as ginger ale and tea.Although ginger is consumed all over the world, its cultivation is limited to the tropics and subtropics, with a global production of over 200 000 tonnes of ginger rhizomes per annum [2].In Australia, the ginger industry has a market value of AU $116 million per annum [3], with production primarily based in southeast Queensland.
Globally, ginger production is threatened primarily by rhizome rot or root rot diseases caused by seed and soil-borne pathogens such as Fusarium spp.and Pythium spp., with these being major contributing factors in yield reduction [4][5][6].Fusarium oxysporum strains infect hundreds of plant species, producing symptoms such as wilts and yellows.However, each strain, or formae specialis (f.sp.), is host-specific and is generally named based on the host to which it is pathogenic [7].Fusarium oxysporum f. sp.zingiberi (Foz) is pathogenic against ginger, and is known by the common names Fusarium yellows [4] or Fusarium wilt [8].External symptoms of Fusarium yellows include stunted growth and yellowing and drooping of older leaves, leading to senescence of individual tillers [9].However, it is the internal discolouration and rotting of the rhizomes that is associated with the reduction of ginger rhizome size and quality.Internally, this brown discolouration is observed in the water-conducting regions of the rhizome and rotting of the tissues in the rhizome cortex [9].
Once the soil is infected, Foz is thought to survive as chlamydopores in the absence of a susceptible ginger host and hence can persist for years in the form of these resting spores in the soil [10].The inoculum concentrations in this study only refer to microconidia.It has not been established if macroconidia or chlamydospores would produce similar results.Chlamydospores, as long-term survival structures, may be the primary source of infection after an extended absence of ginger and it would be particularly beneficial to better understand the role of these other spores in infection cycles.
Commercial production of ginger in Australia relies predominantly on two commercial ginger cultivars, Canton and Queensland [11].However, both cultivars are susceptible to Foz [9,12].The need to address the lack of Foz-and other pathogen-resistant genotypes in the Australian ginger industry [13] is an important priority for research and investment [14].It is therefore important that an efficient method for screening ginger cultivars for resistance to pathogens such as Foz is developed to expedite elite cultivar release.
To study the efficacy of Foz infection, we performed an experiment using five different concentrations of Foz microconidia and analysed the effect of soil spore concentration on disease development in the ginger cultivar Canton, which is the main culinary cultivar produced in Australia [9].The impact of Foz infection on ginger plants was apparent, with symptoms including leaf yellowing and reduced rhizome weight.Foz was detected on plants subjected to as little as 10 microconidia g −1 soil.Symptom severity was positively associated with the amount of spores in the inoculum and negatively associated with plant development.The outcome from this study contributes to our understanding of the threshold levels of Foz required to cause an outbreak of Fusarium yellows in the field as well as provide an optimized methodology for screening ginger genotypes for Foz resistance as new cultivars are developed in the future.

METHODS
Four Fusarium oxysporum strains (UQ6848, UQ6851, UQ6852 and UQ6853) were isolated from ginger plants symptomatic for yellows from the following locations in southern Queensland, Australia: Gympie (one), Bundaberg (one) and Nambour (two).Formae specialis of these isolates were confirmed to be Fusarium oxysporum f. sp.zingiberi based on their ability to cause Fusarium yellows in ginger pot trials and by phylogenetic analysis based on the translation elongation factor 1-alpha (TEF-1α) gene of the isolates [15].Monoconidial isolates were subsequently reisolated using hyphal tips of single germinated conidia.Spore suspensions were prepared for the four isolates according to a previously published method [9] and then mixed in equal ratios of spore concentrations prior to plant inoculation.The isolates were mixed in equal proportions to allow for differences in virulence between isolates.To generate the spore suspensions, flasks of potato dextrose broth were inoculated with each of the isolates and were incubated for 7 days at 26 °C with shaking at 120 r.p.m. on a rotating platform (Ratek, Victoria, Australia).The spores were then extracted, counted and adjusted using sterile water to 10 2 , 10 4 , 10 6 , 10 7 and 10 8 microconidia ml −1 for inoculation on ginger plants, as per a previous study [9].DNA was extracted from the mycelia of the four isolates using a microwave method [16].TEF-1α was PCR-amplified using published primers (5′-ATGGGTAAGGA RGACAAGAC) and (5′-GGAR GTAC CAGT SATC ATGTT) [15] and DreamTaq (Thermo Fisher Scientific, Waltham, MA, USA).A single product of 656 bp was visualized on a 1 % agarose gel and then purified using a

Impact Statement
Phenotypic screening of plants is required for the identification of host resistance against agronomically important soil-borne pathogens.In the case of Foz, a reliable phenotyping method needs to account for environmental variations as well as the sporadic expression of Foz symptoms.By establishing a consistent methodology for optimal parameters for plant growth, we intended to determine the level of inoculum dose required to induce uniform Foz infection.The outcomes of this study will aid future research into ginger infection by Foz and help us to understand the threshold level of Foz that is required to cause an outbreak in the field, potentially contributing to an integrated approach to manage this important disease.
GeneJET PCR purification kit (Thermo Fisher Scientific, Waltham, MA, USA), and Sanger-sequenced at the Australian Genome Research Facility, Melbourne, Australia.
TEF-1α sequences representing 156 isolates belonging to four Fusarium species complexes were retrieved from previous studies [9,15,17,18].Geneious Prime v 2023.0.1 (Biomatter Pty Ltd, Auckland, New Zealand) was used for the phylogenetic reconstruction of the isolates.Firstly, multiple sequence alignment was performed using MAFFT v 7.490 [19].The subsequent alignment was used to pick regions with minimized gaps and derive a consensus alignment sequence of 543 bp, which was then used as an input in MrBayes v 3.2.6 to reconstruct the phylogeny using the Bayesian inference method [20].The running parameters used the GTR-G-I model of substitution with two independent analyses on four Markov chain Monte Carlo (MCMC) chains for 2 000 000 generations.A burn-in rate of 25 % was used to sample every 1000 generations.Fusarium delphinoides (NRRL36160) was used as an outgroup to anchor the phylogenetic tree.The tree branches were transformed into a cladogram and visualized in Geneious Prime.
The ginger plantlets of cultivar Canton were produced by in vitro tissue culture [21,22] and then grown in a glasshouse to maintain a disease-free state at the Maroochy Research Facility, Queensland, Australia.A quantity of approximately 400 g of a premium potting mix (Searles, Kilcoy, Queensland, Australia) was used per 100 mm (L) × 100 mm (W) × 120 mm (D) square pot.In total, 53 plants of the susceptible ginger cultivar Canton at a stem height of 15-20 cm carrying 8-12 leaves each were transferred to a Conviron GEN1000 TA plant growth chamber (Winnipeg, MB, Canada) at the University of Queensland, St Lucia, Australia.The growth parameters include a 14 h light/10 h night photoperiod regime with lights set to 100 % intensity at a photon irradiance rate of 700 µmol m −² s −1 , and day and night temperatures kept at 27 and 24 °C, respectively.Watering was done three times a week.Plants were climatized in the chamber for 1 week.For each pot, 40 ml of a concentrated spore suspension was mixed with 400 g of soil and the plant was potted into the inoculated soil.The final spore concentrations were 10 1 , 10 3 , 10 5 and 10 7 microconidia g −1 of soil, respectively.Control plants were mock inoculated with sterile water.All but the 10 7 microconidia g −1 of the soil treatment group had 10 plants each.For the 10 7 microconidia g −1 of the soil treatment group, due to a limited availability of plants, only three plants were used.Each pot was self-contained in a zip-lock bag and grouped with replicates.External and internal symptoms were assessed 8 weeks post-inoculation.Interval symptom was scored using a 1-6 severity scale based on the visual identification of discoloured regions in the rhizome (Fig. 1).Reisolation of Foz in the rhizome, roots and tillers was performed according to a previous study [9].
The statistical software SPSS v28.0.1.0(142) (IBM Corp., Armonk, NY, USA) was used to perform one-way analysis of variance (ANOVA) in a pair-wise manner, with measurements for the various traits set as dependent variables and the treatments as the categorical variable.Waller-Duncan's multiple range testing was performed as a post-hoc test to separate the means into subsets by least significant difference (LSD).The harmonic mean sample size of 7.2 was estimated and used to account for the unequal variance associated with the uneven sample size (n) of the treatments.The type 1/type 2 error seriousness ratio (k-ratio) was set to 100 (α=0.05).

RESULTS AND DISCUSSION
TEF-1α sequencing showed that UQ6848 (NCBI GenBank: OR425153), UQ6851 (OR425154), UQ6852 (OR425155) and UQ6853 (OR425156) shared 100 % nucleotide identity with each other, as well as with three other known Foz isolates (Foz Goomboorian, Foz Eumundi, Foz BRIP44986).Their sequences differ from that of Foz BRIP39299 by three SNPs and a single base INDEL.The phylogenetic tree reconstructed using Bayesian inference produced a topology that separated the four species complexes of Fusarium (Fig. 2).This phylogeny was presented, and its topology described in a previous study [18].Within the Fusarium oxysporum species complex (FOSC), all four isolates used in this study are clustered together in a subclade with known Foz isolates, including Foz Goomboorian, Foz Eumundi, Foz BRIP44986 and Foz BRIP39299 (Fig. 2).The sequences and the phylogenetic positions of these four isolates within FOSC confirmed their Foz identity.
Inoculum concentration had a discernible effect on the severity of Fusarium yellows in ginger cultivar Canton (Fig. 3).Fozinoculated plants could be easily differentiated from uninoculated controls by the development of yellowing symptoms typical of Foz infection (Fig. 3a).The degree of senescing leaves and tillers was positively associated with the inoculum dosage (Fig. 3b), with sporadic yellowing of stems and leaves observed at the low-to-medium inoculum concentration (10 1 , 10 3 , 10 5 microconidia g −1 soil) in comparison to the uninoculated controls which showed little-to-no yellows.Foz susceptibility was exacerbated at the high inoculum levels (10 6 , 10 7 microconidia g −1 soil), where tiller abortions due to Fusarium yellows occurred at a relatively high rate and the whole root systems appeared discoloured (Fig. 3b).
Internally, rhizome discolouration was positively and quantitatively associated with the inoculum level (Fig. 4a).While the uninoculated controls were completely clean, the 10 1 microconidia g −1 of soil dose produced a low but significant level discolouration with a mean score of 2 (<10 %).This difference is also associated with a significant reduction in rhizome size but not with root length or number of tillers alive (Fig. 4b-d).This suggests that a low dosage of Foz spores can still infect the rhizome, potentially reducing the crop yield despite plants inoculated at low spore concentrations having a similar number of tillers or length of root systems as uninoculated controls.The rhizome discolouration index (RDI) provided the best sensitivity for differentiating the dose levels amongst the four traits scored (Fig. 4a).RDI differentiated the low dose 10 1 microconidia g −1 soil from that of control, as well as differentiating medium doses of 10 3 /10 5 microconidia g −1 soil from the high doses 10 6 /10 7 microconidia g −1 soil.This quantitative effect of discolouration is negatively associated with a reduction in root length and rhizome weight (Fig. 4b).However, rhizome weight seems to be most sensitive to Foz infection, with 10 3 , 10 5 , 10 6 , 10 7 microconidia g −1 soil treatments all suffering similar weight reductions (Fig. 4c).
Furthermore, reisolation of Foz showed that it was present in the rhizome of all treatment groups and in the roots of all except the plants subjected to the spore concentration of 10 1 microconidia g −1 soil (Table 1).The alive tillers count was the least affected by Foz, with a significant reduction only observed in 10 6 microconidia g −1 soil or higher dosage (Fig. 4d).This is consistent with the presence of Foz detected in the tillers of treatment groups 10 6 and 10 7 microconidia g −1 soil but not in the other treatments or the control (Table 1).These observations demonstrate that the external symptoms above ground may not necessarily reflect the reduction in rhizomes and roots below ground at low Foz doses.At the high doses of 10 6 and 10 7 microconidia g −1 soil, the saturation of spores in the soil allowed plants to be uniformly infected, leading to the wilting of whole plants or even plant death.Evaluation methods for screening Fusarium wilt resistance have been available for other plant species such as cotton and lettuce [3,23], but the absence of a Fusarium screening method for ginger has hampered disease research.A previous study also demonstrated that the spore concentrations of fungal pathogens have an effect on the wilt symptoms of ginger [8].In comparison, the data generated in this study show that ginger can respond to Foz at a lower or higher dose than 10 3 -10 5 microconidia g −1 soil, allowing sensitivity in host responses against Foz to be refined.Fig. 2. Determination of the phylogenetic positions of Foz isolates UQ6848, UQ6851, UQ6852 and UQ6853 within the Fusarium oxysporum species complex using translation elongation factor 1-alpha (TEF-1α).The four isolates are highlighted in red.The four Fusarium species complexes presented in this Bayesian inference phylogeny include the Fusarium incarnatum-equiseti species complex (FIESC); the Fusarium fujikuroi species complex (FFSC); the Fusarium oxysporum species complex (FOSC); and the Fusarium solani species complex (FSSC).Where it is known, the formae speciales of pathogenic isolates within FOSC are indicated with a red circle.Branch labels indicate the posterior probability as a percentage.A scale range of 0.05 is indicated below the tree.Management of soil-borne pathogens is a significant challenge for ginger production in Australia, with diseases such as Fusarium yellows, Pythium rot caused by Pythium myriotylum [6] and root-knot nematodes (Meloidogyne incognita and M. javanica) [24,25] requiring a combination of management techniques, including fallowing land, improving soil health, managing drainage and the use of biological control agents and clean rhizomes for planting [26].The application of biocontrol control agents such as Trichoderma has the potential to control and reduce Fusarium spp.and other soil phytopathogens [27].One of the key aspects of controlling Fusarium yellows is the improvements in minimizing the inoculum pressure for diseases in the field by using clean planting materials and schemes through tissue culture [28] or using material produced from clean mother blocks.In Australia, ginger is produced mainly on red ferrosol soils and is managed intensively with inputs and tillage during multiple annual production cycles before the field is rotated to fallow crops [3,29,30].When disease outbreaks occur, it is important to understand the infection process and disease threshold so that Foz can be diagnosed and monitored through immunological, phylogenetic and pathogenicity-related approaches [31].
This study has developed a rigorous methodology to test the virulence of Foz isolates on ginger plants by establishing a minimum threshold level of microconidia per gram of soil required for infection in a controlled environment.This screening method can be used to assess Foz resistance levels in wild relatives and cultivars of ginger.Rhizome discolouration offers a reliable trait marker for screening ginger's susceptibility to Foz.Therefore, it can be used to rapidly fast track elite resistance phenotypes for introduction into commercial ginger cultivars.Further optimizations can potentially allow this method to be adapted to screen for other plant-soil phytopathogen interactions.
Plant breeding for disease resistance is a part of plant disease management.Integrated management practices, including improvements in soil health, addition of organic amendments such as poultry manure and sawdust, annual rotations of suitable cover crops and prolonged fallow between ginger cultivations will help reduce but not completely eliminate Foz in the soil [29,30].Even with management practices applied, re-emergence of the disease can potentially override biological suppression developed through improved soil health [3].It is therefore important to ensure, where possible, that cropping soil used for ginger is not contaminated with soil-borne pathogens such as Foz, as it has been demonstrated that even low numbers of microconidia per gram of soil can still result in significant loss of rhizome quality and quantity, and therefore crop yield.

Funding information
The research conducted in this study was funded by the University of Queensland through a Graduate Research Project (AGRC7618).
Table 1.Three plants randomly selected from each treatment group (n=10, labelled A to J for each treatment from 10 1 to 10 6 , n=3 for 10 7 ) were used to reisolate Foz from the rhizome, roots and tillers.'+' indicates a positive colony that conformed to a typical F. oxysporum f. sp.zingiberi morphology [9,32]  Response: We changed that to the following: ' A previous study also demonstrated that the spore concentration of fungal pathogens have an effect on the wilt symptoms of ginger.' Line 179: 'Reference 15' -Recent studies with positive outcomes related to use of Trichoderma-based BCAs in temperate areas can be added here.

Check reference 7
Response: Added a sentence to discuss the potential use of biological control agents against soil phytopathogens such as Fusarium.
'The application of a biocontrol control agent such as Trichodermahas the potential to control and reduce Fusariumspp.and other soil phytopathogens [27].' 27Tyśkiewicz R, Nowak A, Ozimek E, Jaroszuk-Ściseł J. Trichoderma: The current status of its application in agriculture for the biocontrol of fungal phytopathogens and stimulation of plant growth.Int J Mol Sci2022;23:2329.https:// doi.org/ 10. 3390/ ijms23042329 Please rate the manuscript for methodological rigour If this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?
Reviewer 2: Yes: Reviewer 2 Comments to Author: This manuscript determines the infection threshold and dose-associated symptoms of Fusarium oxysporum f. sp.Zingiberi in the ginger cultivar "Canton", which is of importance in Australia.The research objectives are clearly stated, although to be completely met further cultivars would be included in the experimental design.Notwithstanding, the provided methods and results could be of help for applying in other cultivars.Methods are adequately described to reproduce the experiments once the minor suggestions below are implemented.Identification of Foz isolates by molecular methods would be required, at least to confirm the species.Statistical analysis was appropriate.In general, the article is well written and conclusions are sound by the presented results.Below, my suggestions for improvement of the manuscript are found: 1. Methodological rigour, reproducibility and availability of underlying data -In the abstract, authors declare that their research objectives were: i) "to establish a minimum threshold level of spores per gram of soil required for plant infection" (lines 36-37) ii) "to develop and evaluate a pot inoculation method for suitability in genetic studies on Foz resistance" (lines 37-38) In my opinion, i) has been met, but ii) was only partially covered by the experiment, since for that aim, different cultivars with variable resistance to Foz should be included and compared.

Response:
We would like to clarify that the objective ii) was meant to develop a suitable screening methodology in a controlled environment that can be used to screen for varietal differences in resistance in the future.We have not performed any resistance screen in this study so therefore we have reworded the second objective to: '..to develop and evaluate a pot inoculation method for suitability in screening large number of plants in a controlled environment.' -Line 83: authors state: "To study the efficacy of Foz infection, we performed a replicated experiment using five different concentrations of Foz microconidia", but indeed it is not indicated if the experiment was replicated and results reproducible.Please, add clarification.
Apologies for the confusion.It is a single experiment to determine the Foz dosage response.We have removed the word 'replicated' .However, this is an established method has worked well in disease assays and gene expression analysis on ginger plants.See our publication [9].
-Lines 96-98: authors state that "Formae specialis of these isolates were confirmed to be Fusarium oxysporum f. sp.zingiberi based on their ability to cause Fusarium yellows in ginger pot trials".Why didn't authors complement the identification with a more accurate method to determine whether those isolates are classified as F. oxysporum f. sp.Zingiberi, for example, using molecular markers?It would be required to identify molecularly the isolates at species level.

Response:
We have since performed TEF-1α sequencing on these 4 isolates and created a Bayesian inference phylogenetic analysis using isolates belonging to different Fusarium species complexes to show that they are most likely Foz.
TEF-1α sequences representing 156 isolates belonging to four Fusariumspecies complexes were retrieved from previous studies [9,15,17,18].Geneious Prime v 2023.0.1 (Biomatter Pty. Ltd., Auckland, New Zealand) was used for the phylogenetic reconstruction of the isolates.Firstly, multiple sequence alignment was performed using MAFFT v 7.490 [19].The subsequent alignment was used to pick regions with minimised gaps and derive a consensus alignment sequence of 543 bp which was then used as an input in MrBayes v 3.2.6 to reconstruct the phylogeny using the Bayesian inference method [20].The running parameters used GTR-G-I model of substitution with two independent analyses on four Markov Chain Monte Carlo (MCMC) chains for 2,000,000 generations.A burn-in rate of 25% was used to sample every 1000 generations.F. delphinoides(NRRL36160) was used as an outgroup to anchor the phylogenetic tree.The tree branches were transformed into a cladogram and visualised in Geneious Prime.' Line 192-202 (results) 'TEF-1αsequencing showed that UQ6848 (NCBI-GenBank:OR425153), UQ6851 (OR425154), UQ6852 (OR425155) and UQ6853 (OR425156) shared a 100% nucleotide identity with each other, as well as with three other known Fozisolates (FozGoomboorian, FozEumundi, FozBRIP44986).Their sequences differ to that of FozBRIP39299 by 3 SNPs and a single base INDEL.The phylogenetic tree reconstructed using Bayesian inference produced a topology that separated the four species complexes of Fusarium (Figure 2).This phylogeny was presented, and its topology described in a previous study [18].Within the Fusarium oxysporumspecies complex (FOSC), all four isolates used in this study are clustered together in a subclade with known Fozisolates including FozGoomboorian, FozEumundi, FozBRIP44986 and FozBRIP39299 (Figure 2).The sequence and the phylogenetic positions of these four isolates within FOSC confirmed their Fozidentity.' -Lines 100-101: Could authors explain the reason why they mixed 4 four isolates for the inoculum?
Response: We added the following sentence to explain this.'The isolates were mixed in equal proportions to allow for differences in virulence between isolates.' -Lines 104-105 and rest of manuscript: My suggestion is to use the unit microconidia/mL or microconidia/g of soil, instead of cfu/mL or cfu/g.For the second, it would be required having a probe that the microconidia had the ability to form colonies (for example, by plating diluted inoculum and counting colonies).Please, strict to this unit in the whole text.

Response:
We have changed the unit to microconidia/mL for this and the rest of the document.
-Lines 116-120: Please, rewrite these sentences to clarify how soil inoculation was conducted.As it is explained, it seems that pots with soil were inoculated and then mixed with extra 400 g of soil.
Response: We have rewritten this section as the following: 'For each pot, 40mL of a concentrated spore suspension was mixed with 400g of soil and the plant was potted into the inoculated soil.The final spore concentrations were 10 1 , 10 3 , 10 5 and 10 7 microconidia/g of soil, respectively.' -Line 85: Please, add a reference.-Discussion: Much information about agricultural practises related to ginger are explained, but I miss further discussion on their relation with this study, its importance, and conclusions; mainly focused on the improvements on disease control and management of crop productivity.Also, it would be interesting to discuss limitations of the study, for example: plant variety selection, number of plants, isolate virulence, etc.

Date report received: 30 April 2023 Recommendation: Major Revision
Comments: Lines 1-3: Lowest may refer that 10 cfu/gm soil be the least and there may be a need to run logistic or other growth models to showcase the clearcut point where the threshold is significant.Anything

Please rate the quality of the presentation and structure of the manuscript Good
To what extent are the conclusions supported by the data?Strongly support

Is there a potential financial or other conflict of interest between yourself and the author(s)? No
If this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?Yes SciScore report https://doi.org/10.1099/acmi.0.000605.v1.1 © 2023 The Authors.This is an open-access article report distributed under the terms of the Creative Commons License.

Fig. 1 .
Fig. 1.Ginger rhizome discolouration index used to score the internal symptoms of the diseased plants in this study.The scale was (a) completely clean, no discolouration observed; (b) less than 10 % discolouration including edge effects or small patches, where it is not completely clean; (c) brown discoloured areas occupying over 10 % and up to 25 % of the rhizome; (d) brown discoloured areas occupying over 25 % and up to 50 % of the rhizome; (e) brown discoloured areas occupying over 50 % and up to 75 % of the rhizome; (f) brown discoloured areas occupying over 75 % and up to 100 % of the rhizome.Arrows indicate the visible regions of discolouration in the rhizomes.

Fig. 3 .
Fig. 3.A qualitative assessment of external symptoms of Fusarium yellows on Foz-susceptible ginger cultivar Canton.Plants were examined at 8 weeks post-inoculation.(a) Potted ginger cultivar Canton plants at the time of harvest.(b) Individual representative plants from each treatment with above and below ground level portions cleaned and then visualized.
and '−' indicates the absence of any F. oxysporum-like colonies A previous study' -Doesnt seem to be a reference to the same pathogen, please clarify

Reviewer 2 :
SatisfactoryPlease rate the quality of the presentation and structure of the manuscript Reviewer 2: Good To what extent are the conclusions supported by the data?Reviewer 2: Strongly support Do you have any concerns of possible image manipulation, plagiarism or any other unethical practices?Reviewer 2: No: Please rate the quality of the presentation and structure of the manuscript GoodTo what extent are the conclusions supported by the data?Strongly supportDo you have any concerns of possible image manipulation, plagiarism or any other unethical practices?NoIs there a potential financial or other conflict of interest between yourself and the author(s)?NoIf this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?YesReviewer 1 recommendation and comments https://doi.org/10.1099/acmi.0.000605.v1.4 © 2023 Anonymous.This is an open access peer review report distributed under the terms of the Creative Commons Attribution License.